Bioassays

Malaria

Malaria is among the most life-threatening and widespread diseases in the world, causing 250-300 million cases and about 2 million deaths annually. The disease is caused by four Plasmodium species (i.e. P. falciparum, P. vivax, P. ovale and P. malariae) which are transmitted to humans during the bite of the female anopheles mosquito.

The methodology to test our extract and pure compounds is efficient and accurate for the detection of anti-malarial agents based upon the intercalation of the fluorochrome PicoGreen® into Plasmodium DNA. PicoGreen® is an ultra sensitive fluorescent nucleic acid stain for measuring double-stranded DNA (dsDNA) in solution and enables the detection of quantities as low as 25 pg/mL of dsDNA with a moderately priced spectrofluorometer using fluorescein excitation and emission wavelengths.

This non-radioactive DNA-based assay, using PicoGreen® with 485 nm excitation and 528 nm detection, was developed in Dr. Ortega-Barria's laboratory to measure the growth of Plasmodium falciparum (W2 strain), in erythrocytes. Parasite growth is measured after cells have been incubated with extracts at 50, 10, or 2 microg mL-1 for two days. Chloroquine is used as a positive control.

This methodology has several advantages over the radioactive bioassay used commonly.

-It is as sensitive as the radioactive method
-It is less costly than the radioactive method
-Does not require special waste disposal

Chagas' disease

We screen extracts against a ß-galactosidase-expressing transgenic Trypanosoma cruzi (Tulahuen C4 strain).

Amastigotes and trypomastigotes, the intracellular forms of T. cruzi, , are responsible for the development of Chagas disease or trypanosomiasis americana, and  are the likely target for medication against the disease. In our bioassay T. cruzi parasites are grown intracellularly in Vero cells. For both the intracellular assay and extracellular assay (with axenic epimastigotes), cells are cultured for three days with 50 and 10 µg/mLof the sample to be screened for its antiparasitic activity. Nifurtimox is used as a positive control. T. cruzi growth is determined from the cleavage of Chlorophenol red-ß-D-Galactopyranoside (CPRG) and the color of the metabolite is read at 570nm in a fluorimeter.

Quick facts of Chagas disease:

-Endemic in 21 countries.
-20 million infected / 10-20% mortality
-100 million at risk
-Latin America cost from early death=8.2 billion/year
-100,000 infected in the US.
-No new drugs:
Nifurtimox     (1965)          
Beznidazole   (1971)
-Drugs only active during acute phase (approximately first 30 days)
-Variable response to the drugs. Highly toxic (40-70%)

Leishmaniasis

A novel colorimetric assay was developed and standardized by former AP2 coordinator, Dr. Luz Romero which provided a quantitative and efficient method for the evaluation of anti-leishmanial activity in plant extracts and purified compounds. The assay employs the tetrazolium salt, sodium-2, 3-bis-[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT). In the presence of the electron-coupling agent, phenazine methasulfate, XTT is reduced by mithocondrial dehydrogenase to a water-soluble product in the culture media supernatant whose concentration can be determined by measuring the optical density at 450 nM in a microplate reader.

Optical density values obtained at 450 nM with the XTT method correlated well (r = 0.965) with parasite numbers in a growth curve of L. mexicana, as determined by microscopic count. The minimal number of parasites detected over the control background was 977 promastigotes. The IC50 values obtained for the reference anti-leishmanial drugs, amphotericin-B, hexadecylphosphocholine (Miltefosine) and Ketoconazole were 0.037 g/mL, 10.2 g/mL and 0.023 g/mL, respectively, and were similar to those previously reported.

Cancer

Cell lines studied: MCF-7 (Breast), SF-268 (CNS), NCI-H460 (Lung)